As a 14-year-old De Rosario rejected an offer from AC Milan after a successful trial due to him not being ready to commit to living in Italy.[8] He began his professional career in 1997 at the age of 18, signing with the Toronto Lynx of the A-League. Halfway through the season, however, De Rosario opted to change clubs, signing with German side FSV Zwickau. After two seasons with Zwickau, De Rosario opted to return to North America, signing with the Richmond Kickers in 1999. After a slow 1999 season, in which he registered two goals and five assists, De Rosario exploded in 2000, contributing 15 goals and five assists while leading the team to a 20–6–1 record.

De Rosario before a pre-friendly game with San Jose Earthquakes in 2004

The next season, when Canadian Frank Yallop was named head coach of the San Jose Earthquakes, De Rosario was one of his first acquisitions. De Rosario proved Yallop's judgment right, scoring five goals and four assists in only 1,072 minutes for the Earthquakes in 2001, playing an important role as the team went on to win the MLS Cup; he scored the golden goal in the final and was named MLS Cup MVP. De Rosario had similar success in 2002, registering four goals and eight assists in 1,637 minutes, though the Quakes fell short of a repeat. In 2003, a torn ACL hobbled De Rosario for much of the season but he still managed to make a late surge, registering four goals and three assists in only 686 minutes and helping lead the team to a second MLS Cup championship. De Rosario played 1,214 minutes in 2004, scoring five goals, including the 2004 MLS Goal of the Year, and three assists.

In December 2004 De Rosario had a trial with Nottingham Forest,[9] but he was not offered a contract by the team.

In 2005, following Landon Donovan's departure, De Rosario moved to midfield and promptly led MLS in assists with 13, while scoring nine goals, including the 2005 MLS Goal of the Year – the only player ever to receive that honour in two consecutive years – for a powerful bending free kick in the last regular season game against the Los Angeles Galaxy. He was named to the MLS Best XI.

Due to San Jose's failure to reach a stadium agreement with AEG, De Rosario, along with the rest of his Earthquakes teammates, moved to Houston for the 2006 season. During the 2006 MLS All-Star Game in Chicago, De Rosario scored the only goal of the game in the 70th minute to lift the MLS All Stars to a 1–0 win over Chelsea, a pre-season friendly for the London club. De Rosario was one of only four players on the MLS team to play the entire match.

De Rosario and the Houston Dynamo captured the 2006 MLS Cup title by beating the New England Revolution on November 12, 2006. The Dynamo won in a shootout, and De Rosario successfully converted his penalty kick. De Rosario signed a contract extension with Houston through 2010, where he was reported to make $325,000 per year. He was later transferred to Toronto before the end of his contract.[10]

The next year, De Rosario and the Dynamo repeated the feat by winning the 2007 MLS Cup, beating New England 2–1. De Rosario was named MLS Cup MVP, the first player ever to win the award twice.

De Rosario made his third consecutive all-star appearance at the 2008 MLS All-Star Game in his home country, when the game was held in Toronto. He scored the decisive goal on a penalty kick in the 69th minute in the MLS All-Stars' 3–2 victory over West Ham United.

De Rosario was traded to Toronto FC on December 12, 2008 in return for Julius James and allocation money, after long speculation that De Rosario would move to his hometown club.[11] He made his competitive debut for Toronto FC on Saturday, March 21 against the Kansas City Wizards, setting up Jim Brennan for Toronto's first goal in a 3–2 victory. He scored his first goal for Toronto from a header in a 1–1 draw at BMO Field against FC Dallas.

De Rosario was expected to miss the first two to four weeks of Toronto FC's training camp due to a calf injury obtained in the January 31, 2010 match against Jamaica,[12] and returned to game action in Toronto's 1–0 preseason win versus the University of South Florida.[13]

On April 15, 2010, De Rosario scored twice in Toronto's 2–1 home opener win against the Philadelphia Union.[16] In his next game versus the Colorado Rapids, De Rosario scored his fourth goal of the season, and more importantly, became Toronto FC's all-time leading scorer in the regular season.[17] De Rosario again found the back of the net on April 25 in a 2–0 home win against Seattle Sounders FC, scoring the first goal, his fifth of the season. Until O'Brian White scored the second goal for Toronto, De Rosario had previously scored all of Toronto's goals up until that point in the season.[18] For his efforts in that game, he was awarded the MLS Player of the Week for week 5.[19] De Rosario was again honoured with the Player of the Week award on week 10 of the MLS season, for his two-goal performance against his former team the San Jose Earthquakes. De Rosario had scored the second and third goal in Toronto FC's 3–1 win.[20]

De Rosario continued to have a successful 2010 season for Toronto, culminating in a spot on the MLS All-Star team, scoring a goal in the 5–2 loss to Manchester United.[21]

On December 28, De Rosario was confirmed to be on trial with Scottish Premier League club Celtic by manager Neil Lennon.[23] Dwayne and Celtic inquired about the possibility of a short term loan deal until the MLS season kicked off in March, however new Toronto FC coach Aron Winter and the league denied any further negotiations.[24]

De Rosario scored the first Toronto goal of the 2011 season on March 19 in a 4–2 away defeat to Vancouver Whitecaps FC in what was the league's first all Canadian match up. The goal scored in the 20th minute was also the 8000th goal scored in Major League Soccer's history.[25]

D.C. United acquired De Rosario on June 27, 2011, in exchange for midfielder Dax McCarty.[28] He scored his first goal for the club against his former club New York Red Bulls on his return to Red Bull Arena. On July 30, 2011 his double gave United a 2–0 victory over former club, San Jose. On August 6, 2011 he led United to a 3–3 draw against Toronto F.C. with his hat-trick. All three goals were scored while United played with ten men following an early ejection of goalkeeper Bill Hamid. His second hat-trick was recorded on September 25, 2011 as he recorded all three goals in the span of 9 minutes, setting another MLS record.[29] De Rosario completed the season with a total of 16 goals and 12 assists over 32 games. Of those, 13 goals and 7 assists were made during his 17 games with United.[30]

On August 29, 2012, De Rosario scored his 100th MLS goal, in a 2–2 home draw against the Red Bulls. He was the seventh player to reach the milestone.[31]

After having his option declined by DC United, De Rosario returned to Toronto FC on December 18, 2013, after being chosen in the 2013 MLS Re-Entry Draft.[32] He officially signed with the club on January 9, 2014.[33] It was announced on December 3, 2014 that Toronto had declined the option to renew De Rosario's contract.[34]

In 2007, De Rosario won the male Canadian Player of the Year award making it three consecutive years receiving the honour. In 2007, he scored five goals in eight games, the most in a year for the CMNT since John Catliff in 1993.

De Rosario was picked for his first CONCACAF Gold Cup in four years, after being selected by coach Stephen Hart in late May 2011 for the 23 man tournament roster. After a disappointing, 2–0 defeat to United States in the opening game of the group Canada failed to exit the group with a 1–1–1 record, they only managed to score two goals both from De Rosario at the penalty spot.[36] De Rosario continued his goal scoring for with two goals in the opening stage of 2014 FIFA World Cup qualification, one came against Saint Lucia in early September and the other against Saint Kitts and Nevis in mid November. His goal against Saint Kitts, was his 19th international goal which tied him as Canadian all-time top goal scorer with Dale Mitchell.[37] On December 14, De Rosario was awarded 2011 Canadian Player of the Year receiving 47.7% of the vote, Simeon Jackson in second and Josh Simpson finishing in third. This was the fourth time De Rosario was honoured with the award.[38][39] On September 7, 2012 De Rosario scored his 20th goal for Canada in a 2014 FIFA World Cup qualification match against Panama, making him the all-time leading goal scorer for Canada.[2]

De Rosario's final appearance for Canada was also his last as a player, scoring a goal in a 1–1 draw with Iceland on January 19, 2015.[40]